Preparation of cell extracts, co-immunoprecipitation and immunoblotting were carried out as previously described (Pyronnet et al., 1999 (link)). The antibodies used were as follows: anti-eIF4GI and anti-eIF4GII (gifts of Prof. Nahum Sonenberg); anti-DAP5 (CliniSciences #610742); anti-HA-7 (Sigma); anti-β-tubulin (GeneTex #6288022); anti-4E-BP1, anti-NRF2 and anti-p53 (Cell Signaling Technologies #9452, #12721, and #1C12, respectively); anti-Core 20S (Enzo Life Sciences #PW8155); and anti-NQO1 (Santa Cruz #C19).
Anti 4e bp1
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
Anti-4E-BP1 is a laboratory reagent used to detect and quantify the 4E-BP1 (Eukaryotic Translation Initiation Factor 4E-Binding Protein 1) protein. 4E-BP1 is a translation repressor that binds to and inhibits the eukaryotic translation initiation factor eIF4E, thereby regulating protein synthesis. The Anti-4E-BP1 reagent can be used in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression, localization, and post-translational modifications of the 4E-BP1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti mtor, by Cell Signaling Technology (39 mentions)
Anti phospho 4ebp1, by Cell Signaling Technology (35 mentions)
Anti akt, by Cell Signaling Technology (32 mentions)
Anti p70s6k, by Cell Signaling Technology (28 mentions)
Anti p 4ebp1, by Cell Signaling Technology (25 mentions)
Anti s6, by Cell Signaling Technology (16 mentions)
Anti β actin, by Merck Group (15 mentions)
Anti phospho akt, by Cell Signaling Technology (13 mentions)
Anti phospho mtor, by Cell Signaling Technology (13 mentions)
Anti eif4e, by Cell Signaling Technology (12 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (12 mentions)
Anti phospho 4e bp1 thr37 46, by Cell Signaling Technology (12 mentions)
Anti tsc2, by Cell Signaling Technology (11 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (10 mentions)
Anti eif2α, by Cell Signaling Technology (10 mentions)
Anti parp, by Cell Signaling Technology (10 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (10 mentions)
Anti gapdh, by Cell Signaling Technology (9 mentions)
Anti caspase 3, by Cell Signaling Technology (9 mentions)
Anti p akt, by Cell Signaling Technology (8 mentions)
139 protocols using anti 4e bp1
1
Protein Interaction Analysis by Co-IP
2
Thapsigargin and Rapamycin Induced Cell Stress Response
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Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was from Invitrogen (Carlsbad, CA, USA). DMEM and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The Hoechst kit and Lyso-Tracker Red probe for acidic lysosome staining were from Beyotime (Haimen, Jiangsu, China). Anti-PERK, Anti-BiP, anti-p-eIF2α, anti-eIF2α, anti-cleaved-caspase 3, anti-LC3, anti-p-AKT, anti-p-p70S6K1, anti-p70S6K1, anti-p-4EBP1 and anti-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ATF-6, anti-p62 and anti-GAPDH were from Abcam (Cambridge, UK). The CHOP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were obtained from Sigma-Aldrich with the highest purity available.
3
Western Blot Analysis of Protein Targets
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Cells were washed twice with PBS, and lysed with RIPA buffer containing 0.1 % SDS, 10 μg aprotinin/mL, 100 μg PMSF/mL and 1 % phosphatase inhibitor cocktail (Sigma). Cell lysates were subjected to SDS-PAGE, and the resolved proteins were transferred to a PVDF membrane (Millipore). The membrane was blocked with 5 % Block Ace (Yukijirushi) in Tris buffered saline (TBS) containing 0.1 % Tween-20, and was probed with the following primary antibodies: anti-ITAF45 (Santa Cruz Biotechnology), anti-PTB (Cell Signaling), anti-4E-BP1 (Cell Signaling), and anti-βactin (SIGMA)]. The membranes were then washed 3 times with 0.05 % Tween- 20-TBS for 10 min. Secondary antibodies (peroxidase-conjugated anti-rabbit or anti-mouse IgG; DAKO) were subsequently added, and specific protein bands were visualized with enhanced chemical luminescence (GE Healthcare).
4
Immunoblotting Analysis of Signaling Proteins
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We used the following antibodies: anti-Akt (No. 9272), anti-phospho-Akt (Ser473; No. 9271), anti-S6K (No. 9202), anti-phospho-S6K (Thr389; No. 9205), anti-S6 (No. 2217), anti-phospho-S6 (Ser235/236; No. 4858), anti-4EBP1 (No. 9644), anti-phospho-4EBP1 (Thr37/46; No. 2855), anti-GSK3β (No. 12456), anti-phospho-GSK3β (Ser9; No. 5558), anti-Ampk (No. 2532), anti-phospho-Ampk (Ser9; No. 2535), anti-mouse IgG (No. 7076), and anti-rabbit IgG (No. 7074) purchased from Cell Signaling Technology (Massachusetts, United States of America). Anti-α-Tubulin (12G10) was purchased from DSHB (Iowa, United States of America). Anti- Laminin α-2 (SC-59854) was purchased from Santa Cruz Biotechnology (Texas, United States of America).
5
Western Blot Analysis of Protein Lysates
6
Comprehensive Immunoblot Analysis of Inflammatory Signaling
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The following antibodies (Abs) and reagents were used in present studies: anti-IL-1β (human specific, mouse specific, and human & mouse specific), anti-phospho NF-κB, anti-NF-κB, anti-phospho ERK, anti-ERK, anti-phospho p38, anti-p38, anti-phospho 4E-BP1, anti-4E-BP1, anti-phospho Mnk1, anti-Mnk1, anti-phospho eIF4E, anti-eIF4E, anti-phospho MK2, anti-MK2, and anti-streptavidin-HRP antibodies were purchased from cell signaling technology (Danvers, MA). Anti-β-actin, Dimethyl sulfoxide, Actinomycin D, Cyclohexamide, ultrapure E. coli O111:B4 LPS, Rapamycin, PD98059, and SB202190 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-NLRP3 antibody was purchased from Adipogen (San Diego, CA). Torin 1 and FR180204 were purchased from Merck Millipore (Billerica, MA). CGP57380 and MK25 were purchased from TOCRIS (Ellisville, MO, USA) and Cayman Chemical (Ann Arbor, MI), respectively. Recombinant human M-CSF and mouse M-CSF were purchased from R&D system (Minneapolis, MN).
7
Immunoblot Analysis of Cellular Signaling
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Immunoblot analysis was conducted as described previously [48 (link)]. Briefly, equal amounts of proteins were resolved on an SDS-polyacrylamide gradient gel and transferred by electroblotting onto a nitrocellulose membrane. Membranes were probed with the indicated primary antibodies. The specific signals were visualized with a chemiluminescence detection system using appropriate secondary antibodies (Perkin-Elmer, Waltham, MA, USA). The following antibodies were used for immunoblotting: anti-TOP2B (Abcam, Cambridge, MA, USA), anti-HIF-1α, anti-ARNT/HIF-1β (BD Transduction, San Jose, CA, USA), anti-EPAS1/HIF-2α, anti-mTOR, anti-MUC1, anti-HK2, anti-PDK1, anti-4EBP1, anti-phospho-4EBP1 (Ser65), anti-S6, anti-phospho-S6 (Ser235/236), anti-S6K, anti-phospho-S6K (Thr389) and anti-RPS3 (Cell Signaling Technology, Danvers, MA, USA). TOP2B, mTOR and RPS3 were used as controls.
8
Immunohistochemical analysis of apoptosis and autophagy
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Tissue samples were deparaffinized with xylene, and an UltraSensitiveTM SP (Mouse/Rabbit) IHC kit (KIT-9730, MX Biotechnologies) was used for detection. After proteolytic digestion and peroxidase blocking, the tissue specimens were incubated with anti-Caspase-3 (#9664S, Cell Signaling Technology), anti-Cleaved Caspase-3 (#9662, Cell Signaling Technology), anti- SQSTM1 (sc-48402, Santa Cruz), anti-4E-BP1 (#9452, Cell Signaling Technology), anti-Vimentin (A19607, ABclonal), and anti-E-Cadherin (#3195T, Cell Signaling Technology) antibodies at a dilution of 1:200. The biotinylated secondary antibodies in the kit were used according to the manufacturer’s instructions after washing the samples. Then, the samples were washed with PBS three times and then incubated with streptavidin-horseradish peroxidase complex. A DAB substrate kit was used for staining. Liver tissues from control group mice were used as positive and negative control. Negative controls were prepared by substituting with normal rabbit or mouse IgG.19 (link) The immunoreactivity scores were calculated by two pathologists using a system based on multiplying the staining percentage (0: 0–5%, 1: 6–25%, 2: 26–50%, 3: 51–75% and 4: 76–100%) and intensity (0: negative, 1: weak, 2: moderate and 3: strong).
9
Protein Extraction and Western Blot Analysis
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Ice-cold PBS was used to rinse cells, followed by lysis with lysis buffer (50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM EDTA, 0,5% Triton X-100) containing PMSF (1 mM), proteinase inhibitors (Roche, Mannheim, Germany, #1697498) and phosphatase inhibitors (10 mM NaPPi, 200 μM NaVO3, 25 mM NaF). Incubation for 15 min on ice was followed by centrifugation of lysates at 16 000 g for 20 min. Bradford Assays (Bio-Rad, Munich, Germany) were used to measure protein concentrations. For electrophoresis, 20-40 μg of protein was separated with 10-15% polyacrylamid gels and blotted onto nitrocellulose membranes (Bio-Rad) by standard procedures. Membranes where washed, incubated over night with primary antibody, washed again and incubated with secondary antibody (1:3000) coupled to horseradish peroxidase (Bio-Rad). Visualization was performed by an enhanced chemiluminescence detection system (GE Healthcare, Munich, Germany). Following primary antibodies were used: anti-beta-Actin (Sigma, Deisenhofen, Clone AC15, A5441), anti-4ebp1 (Cell Signaling, Boston, #9644), anti-p-4ebp1 (Cell Signaling, #2855), anti-rpS6 (Cell Signaling, #2317), anti-p-rpS6 (Cell Signaling, #4858), anti-p27 (DakoCytomation, Clostrup, Clone SX53G8, M 7203). Everolimus was usually used in concentrations of 1 μM for 72 h, leucine (Sigma-Aldrich, St. Louis, MO) for 2 h at concentrations of 10 mM.
10
Western Blot Assay of Signaling Pathways
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The Western blots were carried out according to the standard protocol. The following primary antibodies were used: phospho‐Akt (Thr308) rabbit mAb (13038), phospho‐Akt rabbit mAb (Ser 473) (4060), anti‐Akt (pan) rabbit mAb (4691), phospho‐P70s6k rabbit mAb (9205), P70s6k rabbit mAb (2708), phospho‐4ebp1 (#13396, monoclonal), anti‐4ebp1 (#9644), anti‐phospho‐B‐Raf (#2696, polyclonal), anti‐B‐Raf (#9433, monoclonal), anti‐phospho‐Erk (#4370, monoclonal), anti‐Erk (#4695, monoclonal) (all from Cell Signaling Technology, Beverly, MA, USA). The band density was quantified using ImageJ software (NIH, Rockville, MD, USA).
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